Bottom Line:
A strong CodY-binding site located downstream of the transcription start point conferred negative regulation by direct interaction with CodY.Additionally, sequences upstream and downstream of the promoter were required for repression by a second pleiotropic B. subtilis regulator, ScoC, whose own expression is repressed by CodY.The increase in BraB expression only at intermediate activities of CodY may facilitate the uptake of BCAA when they are not in excess but prevent unneeded BraB synthesis when other BCAA transporters are active.

Affiliation: Department of Molecular Biology and Microbiology, Tufts University School of Medicine, Boston, Massachusetts, United States of America.

ABSTRACTThe global transcriptional regulator, CodY, binds strongly to the regulatory region of the braB gene, which encodes a Bacillus subtilis branched-chain amino acid (BCAA) permease. However, under conditions that maximize CodY activity, braB expression was similar in wild-type and codY mutant cells. Nonetheless, expression from the braB promoter was significantly elevated in cells containing partially active mutant versions of CodY or in wild-type cells under growth conditions leading to intermediate levels of CodY activity. This novel pattern of regulation was shown to be due to two opposing mechanisms, negative and positive, by which CodY affects braB expression. A strong CodY-binding site located downstream of the transcription start point conferred negative regulation by direct interaction with CodY. Additionally, sequences upstream and downstream of the promoter were required for repression by a second pleiotropic B. subtilis regulator, ScoC, whose own expression is repressed by CodY. ScoC-mediated repression of braB in codY mutants cells was as efficient as direct, CodY-mediated repression in wild-type cells under conditions of high CodY activity. However, under conditions of reduced CodY activity, CodY-mediated repression was relieved to a greater extent than ScoC-mediated repression was increased, leading to elevated braB expression. We conclude that restricting increased expression of braB to conditions of moderate nutrient limitation is the raison d'être of the feed-forward regulatory loop formed by CodY and ScoC at the braB promoter. The increase in BraB expression only at intermediate activities of CodY may facilitate the uptake of BCAA when they are not in excess but prevent unneeded BraB synthesis when other BCAA transporters are active.

pgen.1005600.g008: Competition between CodY and ScoC for braB binding.A and B. The braB64 DNA fragment, obtained by PCR with oligonucleotides oBB730 and oBB731 and labelled on the template strand, was incubated for 32 min with increasing amounts of purified ScoC (A) or CodY (B) in the presence of 10 mM ILV. C and D. The braB64 DNA fragment was preincubated for 16 min with 12.5 nM CodY (C) or 800 nM ScoC (D), and then increasing concentrations of either ScoC (C) or CodY (D) were added for an additional 16 min. The positions of the free DNA fragment and protein-DNA complexes are indicated by right-pointing and left-pointing arrows, respectively. Protein concentrations used (nM of monomer) are reported below each lane; concentrations corresponding to the apparent KD for binding are underlined.

Mentions:
The downstream CodY- and ScoC-binding sites partly overlap (Fig 1A). To address the possibility that CodY and ScoC compete for binding at this location, we analyzed interaction of these proteins with a short, 64-bp braB fragment, containing CodY-binding site II and ScoC-binding sites II and III (Fig 1B). In accord with the results described above, ScoC bound this fragment in gel shift experiments less efficiently (KD≈150 nM) than did CodY (KD≈5 nM) (Fig 8A and 8B). Nevertheless, ScoC, in a concentration-dependent manner, was able to replace CodY efficiently in a preformed braB-CodY complex as evidenced by formation of ScoC-specific complexes with higher mobility and the decrease in the amount of braB-CodY complexes with lower mobility (Fig 8C). The CodY-mediated displacement of ScoC from the preformed braB-ScoC complex cannot be recognized confidently because of the low mobility of CodY-specific complexes (complexes containing both proteins would have a similar low mobility). However, by comparing and Fig 8B and 8D, it is clear that CodY bound much less efficiently to preformed braB-ScoC complexes than to free braB DNA, confirming competition between the two proteins for binding. A similar competition between CodY and ScoC was previously detected at the oppA promoter [19].

pgen.1005600.g008: Competition between CodY and ScoC for braB binding.A and B. The braB64 DNA fragment, obtained by PCR with oligonucleotides oBB730 and oBB731 and labelled on the template strand, was incubated for 32 min with increasing amounts of purified ScoC (A) or CodY (B) in the presence of 10 mM ILV. C and D. The braB64 DNA fragment was preincubated for 16 min with 12.5 nM CodY (C) or 800 nM ScoC (D), and then increasing concentrations of either ScoC (C) or CodY (D) were added for an additional 16 min. The positions of the free DNA fragment and protein-DNA complexes are indicated by right-pointing and left-pointing arrows, respectively. Protein concentrations used (nM of monomer) are reported below each lane; concentrations corresponding to the apparent KD for binding are underlined.

Mentions:
The downstream CodY- and ScoC-binding sites partly overlap (Fig 1A). To address the possibility that CodY and ScoC compete for binding at this location, we analyzed interaction of these proteins with a short, 64-bp braB fragment, containing CodY-binding site II and ScoC-binding sites II and III (Fig 1B). In accord with the results described above, ScoC bound this fragment in gel shift experiments less efficiently (KD≈150 nM) than did CodY (KD≈5 nM) (Fig 8A and 8B). Nevertheless, ScoC, in a concentration-dependent manner, was able to replace CodY efficiently in a preformed braB-CodY complex as evidenced by formation of ScoC-specific complexes with higher mobility and the decrease in the amount of braB-CodY complexes with lower mobility (Fig 8C). The CodY-mediated displacement of ScoC from the preformed braB-ScoC complex cannot be recognized confidently because of the low mobility of CodY-specific complexes (complexes containing both proteins would have a similar low mobility). However, by comparing and Fig 8B and 8D, it is clear that CodY bound much less efficiently to preformed braB-ScoC complexes than to free braB DNA, confirming competition between the two proteins for binding. A similar competition between CodY and ScoC was previously detected at the oppA promoter [19].

Bottom Line:
A strong CodY-binding site located downstream of the transcription start point conferred negative regulation by direct interaction with CodY.Additionally, sequences upstream and downstream of the promoter were required for repression by a second pleiotropic B. subtilis regulator, ScoC, whose own expression is repressed by CodY.The increase in BraB expression only at intermediate activities of CodY may facilitate the uptake of BCAA when they are not in excess but prevent unneeded BraB synthesis when other BCAA transporters are active.

Affiliation:
Department of Molecular Biology and Microbiology, Tufts University School of Medicine, Boston, Massachusetts, United States of America.

ABSTRACTThe global transcriptional regulator, CodY, binds strongly to the regulatory region of the braB gene, which encodes a Bacillus subtilis branched-chain amino acid (BCAA) permease. However, under conditions that maximize CodY activity, braB expression was similar in wild-type and codY mutant cells. Nonetheless, expression from the braB promoter was significantly elevated in cells containing partially active mutant versions of CodY or in wild-type cells under growth conditions leading to intermediate levels of CodY activity. This novel pattern of regulation was shown to be due to two opposing mechanisms, negative and positive, by which CodY affects braB expression. A strong CodY-binding site located downstream of the transcription start point conferred negative regulation by direct interaction with CodY. Additionally, sequences upstream and downstream of the promoter were required for repression by a second pleiotropic B. subtilis regulator, ScoC, whose own expression is repressed by CodY. ScoC-mediated repression of braB in codY mutants cells was as efficient as direct, CodY-mediated repression in wild-type cells under conditions of high CodY activity. However, under conditions of reduced CodY activity, CodY-mediated repression was relieved to a greater extent than ScoC-mediated repression was increased, leading to elevated braB expression. We conclude that restricting increased expression of braB to conditions of moderate nutrient limitation is the raison d'être of the feed-forward regulatory loop formed by CodY and ScoC at the braB promoter. The increase in BraB expression only at intermediate activities of CodY may facilitate the uptake of BCAA when they are not in excess but prevent unneeded BraB synthesis when other BCAA transporters are active.